Establishment and characterization of cell liens from the walker carcinoma 256 able to grow in suspension culture and deficient in thymidien kinase

Summary A cell line from the Walker carcinosarcoma 256 of the rat has been established in suspension culture in medium with 5% bovien calf serum for over 350 generations, with an average population doubling time of 17 h, a plating efficiency of 56%, a colony forming efficiency of 32%, and a good capacity to form colonies in soft agar. The cells are morphologically indistinguishable from those in the solid tumor and ascites as checked by transmission and scanning electron microscopy. The karyotype is characterized by a modal number of 65 chromosomes and by the presence of a marker metacentric chromosome. The cells express thymidine kinase, λ-glutamyl transpeptidase, and alkaline phosphatase; are aggultinable by concanavalin A; and can be synchronized by the triple thymidine block. They induce primary tumors, both subcutaneously (solid) and intraperitoneally (ascitic), in the rat; are able to metastasize upon injection by the tail vein; and invade the chorioallantoic membrane of the chick embryo. Cells in suspension can be transferred to monolayers, considerably decreasing their tumorigenicity without affecting the other parameters studied, and can be switched back to suspension culture. DNA-mediated transfection showed that DNA from these cells can transform the NIH-3T3 line. Upon growth of the monolayers in a BrdUr-containing medium, a sub-line was establihed that was cloned into a thymidine kinase-deficient line unable to grow, in HAT medium and with properties otherwise similar to those of the parental wild type cells. This work was supported by grants from the “Consejo Nacional de Investigaciones Cientificas y Tecnologicas' (CONICIT) and from the ‘Consejo de Desarrollo Cientifico y Humanistico’ of the Central University of Venezuela.     

Effect of indomethacin in vivo on prostaglandin content of several rabbit tissues

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle in vivo. PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.     

Effect of vasopressin gene expression on the growth of walker 256 carcinosarcoma in rats

The growth pattern of the Walker 256 solid tumor has been studied in rats with different doses of the mutant vasopressin gene. In contrast to the vasopressin gene of normal WAG rats, that of mutant Brattleboro rats has a deletion in the coding region that blocks expression at the translation level. The mutation is inherited as a recessive character and is expressed in homozygous Brattleboro rats as diabetes insipidus with an increased water consumption because of the absence of vasopressin in the blood. (WAG × Brattleboro) F₁ hybrids have the same normal phenotype as WAG rats, including a low water consumption. Walker 256 carcinosarcoma, which is not strain-specific, intensely grows only in WAG and (WAG × Brattleboro) F₁ rats. In these groups, the growth of the tumor leads to the animal death within approximately 30 days after the inoculation of tumor cells. In Brattleboro rats, the carcinosarcoma grows less intensely: the tumor node somewhat increases only within the first two weeks, after which the tumor began to decrease and eventually disappears altogether. Both characters exhibit a 100% concordance at the individual level.     

Effect of prostaglandin inhibition by indomethacin on plasma active and inactive renin concentration in men.

The effect of inhibition of prostaglandin synthesis by indomethacin on active renin and on acid-activable inactive renin was studied in nine healthy, sodium-replete men, both at rest and exercise. These volunteers were investigated after pretreatment with placebo or indomethacin, 150 mg daily for 3 days. Indomethacin induced a decrease in active (p = 0.004), total (p less than 0.001), and inactive (p = 0.02) renin at rest recumbent on average by 42, 19, and 8%, respectively, and at rest sitting on average by 45, 15, and 3%, respectively. Inhibition of prostaglandins with indomethacin reduced (p less than 0.001) active and total renin at each level of work load but not (p = 0.32) inactive renin. However, the exercise-induced stimulation (p less than 0.05) of active and total renin still occur during indomethacin. Indomethacin reduced (p less than 0.001) at rest sitting and at maximal exercise the plasma concentrations of immunoreactive prostaglandins E2 by 50 and 54%, respectively, prostaglandin F2 alpha by 36 and 39%, respectively, and 13,14-dihydro-15-keto-prostaglandin F alpha by 38 and 60%, respectively. The urinary excretion of immunoreactive prostaglandin E2 and F2 alpha was also reduced.     

Ovarian and plasma PGE and PGF levels in naturally ovulating brook trout (Salvelinus fontinalis) and the effects of indomethacin on prostaglandin levels

Plasma and ovarian levels of PGE and PGF were measured by radioimmunoassay in naturally ovulating brook trout ($\text{Salvelinus fontinalis}$). At 12 hours post-ovulation, PGF levels in plasma and ovarian tissue were significantly elevated over levels in gravid controls. Plasma PGF levels were still elevated 5 – 7 days post-ovulation. Indomethacin treatment (10 ug/gm body weight) of ovulatory brook trout significantly decreased plasma and ovarian PGF levels by 12 hours post-ovulation. No significant differences were observed in plasma PGE levels, through gravid controls contained significantly elevated ovarian PGE levels as compared to ovulated fish.     

Effect of alpha-irradiation on sensitive and super resistant mutants of Escherichia coli

A study was made of the sensitivity of E. coli (the wild type, rec A13 and Gamr 444 mutants) to gamma- and alpha-radiation. The most pronounced differences in radiosensitivity of the strains under study were noted with gamma-radiation. The sensitivity of the studied strains to alpha-radiation was levelled. The authors discuss the mechanisms of the effects observed in various E. coli strains exposed to ionizing radiation of different LET.     

Effect of continuous intrauterine administration of prostaglandin F2 alpha and indomethacin on fertilization of rabbits

The effect of continuous intrauterine administration of prostaglandin F2 alpha (PGF2 alpha) or indomethacin or indomethacin together with PGF2 alpha and PGE2 or vehicle on fertilization of rabbits was studied. These substances and vehicle were delivered into the cornua of the uterus via an Alzet minipump for 11 days. The animals were inseminated vaginally. Compared with controls (104 eggs of which 88.5% were fertilized) a reduction of the fertilization rate was observed with indomethacin (74 eggs of which 70% were fertilized). Exogenously added PGF2 alpha did not change the fertilization rate. The administration of indomethacin together with PGE2 raised the fertilization rate to 86% (63 eggs of which 54 were fertilized). The application of PGF2 alpha together with indomethacin showed a fertilization rate of 85% (59 eggs of which 50 were fertilized). The indomethacin application was associated with a reduction of prostaglandin production in several tissues from the female genital tract, showing that indomethacin is taken up by the endometrium of the rabbit. The ovary, oviduct, cervix and vagina were mainly affected by this treatment. The route of transport of this drug is not known, however. The reduction of the total number of eggs together with the decrease of the fertilization rate after indomethacin administration point towards multiregional sites of interference of prostaglandins in reproductive functions. PGF2 alpha seems to be the more important factor since PGE2 may be converted to PGF2 alpha in reproductive tissues.     

Effect of asparaginase on cell membranes of sensitive and resistant mouse lymphoma cells

High concentrations of Escherichia coli asparaginase (80 U/ml) altered the binding of concanavalin A (Con A) to L 5178Y murine lymphoma cells that are sensitive to the cytotoxic action of this enzyme. Incubation of the asparaginase sensitive line in asparagine-free media or media containing Acinetobacter glutaminase-asparaginase did not alter the Con A binding of these cells. Escherichia coli asparaginase had no effect on Con A binding of two asparaginase resistant L5178Y cell lines that were isolated and maintained in asparagine depleted or asparaginase containing medium. The E. coli asparaginase preparation inhibited protein and glycoprotein biosynthesis to comparable degrees. It did not have proteolytic or glycolytic activity. Escherichia coli asparaginase did not alter the binding of wheat germ, soybean or ricin agglutinins to any of these cell lines. These data suggest that high concentrations of E. coli asparaginase have a specific effect on the Con A receptor in the sensitive line.     

Effect of honey bee venom on prostaglandin levels in mouse skin

The effects of honey bee venom on prostaglandin (PG) E levels were studied in mouse skin under and conditions. Levels of PGE were increased 10.8-fold after 15 minutes exposure to reconstituted bee venom and 3.8-fold 35 minutes after a bee sting . Phospholipase A₂ (PLA₂), a major component of bee venom, also caused a 10.9-fold increase in PGE levels and may be primarily responsbiel for this response of skin to bee venom.     

Effect of ibuprofen on menstrual blood prostaglandin levels in dysmenorrheic women

In a randomized crossover study 15 dysmenorrheic women were treated during two consecutive menstrual periods, once with the potent prostaglandin-synthesis inhibitor: ibuprofen and once with an identical looking placebo. Each patient was medicated for 12 hours during the first day of her menstrual flow and was subsequently fitted with a cervical cup for the collection of menstrual blood during three hours. In these samples the concentrations of prostaglandin (PG)F and PGE were measured by radioimmunoassay.The patients receiving placebo had high PGF levels 135 ± 27 ng/ml (Mean ± S.E.) which were significantly reduced by Ibuprofen to 24 ± 5 ng/ml (P<0.001). The PGE concentrations decreased from 5 ± 1 ng/ml to 2 ± 1 ng/ml (P<0.05). Ibuprofen also reduced the menstrual pain significantly (P<0.001). These results substantiate the earlier conclusion that a causal relationship exists between effective treatment with PG-synthesis inhibitors and decrease in menstrual blood PG levels, intrauterine pressure and dysmenorrheic pain.     

Effect of prostaglandin El,E2 or indomethacin on serum parathyroid hormone and calcitonin in the rat

Infusion of 100 or 200 ng/min of Prostaglandin El (PGEl) or of 100 ng/min of PGE2 increased serum parathyroid hormone (PTH) in the rat. These infusions, however, had no significant effect on serum calcitonin (CT). Administration of 10 mg/kg of indomethacin for 3 days had no significant effect on basal serum PTH, CT or Calcium (Ca). EDTA infusion increased serum PTH to a similar degree in the vehicle- or indomethacin-treated rats. Therefore, endogenous prostaglandins do not appear to play a role in the secretion of PTH or CT.     

Effect of indomethacin and prostaglandin E2 on structural differentiation of rat endometrium during artificially induced decidualization

Morphological differentiation of uterine stromal and luminal epithelial cells was studied in steroid-injected ovariectomized rats following unilateral intrauterine instillation of sesame oil, phosphate-buffered saline containing gelatin (PBSG), PBSG + indomethacin (IM; an inhibitor of prostaglandin synthesis), or PBSG + IM + prostaglandin E2 (PGE2). The latter two treatments were preceded by a subcutaneous injection of IM. Uteri were examined by light and electron microscopy at intervals between 8 and 120 hr (n = 4/treatment/time). Differentiation began in the periluminal antimesometrial region and progressed peripherally and towards the mesometrial aspect in all groups. Structural features and timing of differentiation were similar for oil-injected and PBSG-infused uteri. Administration of IM inhibited the onset of the decidual cell reaction and had deleterious effects on the luminal epithelium. Inclusion of PGE2 in the instillate accelerated stromal cell differentiation and overcame the inhibitory effect of IM. The results implicate prostaglandins, particularly PGE2, in endometrial transformation during decidualization.     

Effects of the herbicide isopropyl-N-phenyl carbamate on microtubules and MTOCs in lines of Nicotiana sylvestris resistant and sensitive to its action

To clarify the mechanism of isopropyl-N-phenyl carbamate (IPC) action on higher plant cells the sensitivity of microtubules (cortical network and mitotic arrays) and microtubule organizing centers to IPC treatment (30 microM) in IPC-resistant and sensitive Nicotiana sylvestris lines was studied. It was clearly demonstrated that IPC does not depolymerize plant MTs but causes the MTOC damage in cells, which results in MTOC fragmentation, splitting of the spindle poles and in abnormal division spindle formation. It was also found that IPC-resistance of mutant N. sylvestris line correlates not with tubulin resistance to IPC action but possibly with resistance of one of the proteins involved in MTOC composition.     

Effect of ibuprofen on menstrual blood prostaglandin levels in dysmenorrheic women.

In a randomized crossover study 15 dysmenorrheic women were treated during two consecutive menstrual period, once with the potent prostaglandin-synthesis inhibitor: ibuprofen and once with an identical looking placebo. Each patient was medicated for 12 hours during the first day of her menstrual flow and was subsequently fitted with a cervical cup for the collection of menstrual blood during three hours. In these samples the concentrations of prostaglandin (PG)F and PGE were measured by radioimmunoassay. The patients receiving placebo had high PGF levels 135 +/- 27 ng/ml (Mean +/- S.E.) which were significnatly reduced by Ibuprofen to 24 +/- 5 ng/ml (P less than 0.001). The PGE concentrations decreased from 5 +/- 1 ng/ml to 2 +/- 1 ng/ml (P less than 0.05). Ibuprofen also reduced the menstrual pain significantly (P less than 0.001). These results substantiate the earlier conclusion that a causal relationship exists between effective treatment with PG-synthesis inhibitors and decrease in menstrual blood PG levels, intrauterine pressure and dysmenorrheic pain.     

The effect of triadimenol on the cytology and growth of sensitive and resistant strains of Ustilago avenue

Two triazole resistant mutants of Ustilago avenae and a wild type strain were treated with different concentrations of the fungicide triadimenol in liquid cultures. Growth rates were measured and the morphology of the sporidia examined by light and electron microscopy. After treatment with 1 – 10 μg ml^-1 for 4 days wild type sporidia increased in growth rate, changed from budding to filamentous growth and had considerably thicker cell walls. Their cytoplasm also showed strong degeneration and damage to the cell organelles. The resistant strains were not influenced under these conditions but under longer exposure (14 days) to higher fungicide concentrations (50 to 200 μg ml^-1) similar changes to growth rates, morphology and cell wall thickness also occurred. However, cell disruption only occurred at 200 to 250 μg triadimenol ml^-1 and there were differences in cell wall thickening. One resistant strain (r13) never showed thickening, while in the other (r8), the wall consisted of two distinct layers. The implications of these results are discussed in relation to possible resistance mechanisms.     

Effect of indomethacin, epinephrine, prostaglandin E2 and insulin on lipolysis in bovine adipose tissue in vitro.

1. Fasting of ad libitum- or maintenance-fed steers for 4 to 9 days did not alter basal lipolytic rates in vitro. 2. Epinephrine stimulation of adipose tissue of fasted steers resulted in greater (P less than 0.05) lipolysis than in tissue from fed steers. 3. Prostaglandin E2 (PGE2) did not alter epinephrine-stimulated lipolysis in ad libitum- or maintenance-fed cattle. 4. Indomethacin did not influence basal lipolysis, even in the presence of PGE2. 5. Insulin neither affected basal lipolysis nor inhibited dibutyl cAMP-stimulated lipolysis.     

Effect of 28-homobrassinolide on the performance of sensitive and resistant varieties of Vigna radiata

A study was undertaken to examine the morpho-physiological alterations under different concentrations of 28-homobrassinolide (HBL) in two contrasting varieties of Vigna radiata. Sterilized seeds of V. radiata (T-44 and PDM-139) were inoculated with specific Rhizobium and allowed to grow and then 14 day old seedlings were exposed to different concentrations (0, 10⁻¹⁰, 10⁻⁸, or 10⁻⁶ M) of HBL and allowed to grow under natural environmental conditions. At the 15 and 21 day stage, plants were harvested to evaluate various parameters. Results clearly indicated that growth bio-markers, accumulation of proline and activities of various antioxidant enzymes increased significantly in T-44 at a later stage of growth in the presence of HBL whereas, 10⁻⁸ M showed the most promising response. It is concluded that HBL modifies the physiological functions and biochemical metabolism of V. radiata by increasing photosynthetic efficiency at an early stage of growth and antioxidant system in T-44 at a later stage of plant growth that are manifested in growth at later stages. It is believed that increased accumulation of proline and enhanced antioxidant system provide strength to the plants to withstand environmental cues.